Ion-channels are important therapeutic targets. Neuronal communication, heart function, and memory all critically rely upon the function of ligand-gated and voltage-gated ion-channels. In addition, a broad range of chronic and acute pathophysiological states in many organs such as the heart, gastrointestinal tract, and brain involve ion channels. Indeed, many existing drugs bind receptors directly or indirectly connected to ion-channels. For example, anti-psychotic drugs interact with receptors involved in dopaminergic, serotonergic, cholinergic and glutamatergic neurotransmission.
Voltage clamp methods are superior to any other technology for measuring ion channel activity in cells (see, e.g., Neher and Sakmann, Nature 260: 799-802; Hamill, et al., 1981, Pflugers Arch 391: 85-100; Sakmann and Neher, 1983, In Single-Channel Recording pp. 37-52, Eds. B. Sakmann and E. Neher. New York and London, Plenum Press).
Among voltage clamp techniques, patch clamp is most suitable for measuring currents in the pA range (see e.g. Neher and Sakmann, 1976, supra; Hamill, et al., 1981, supra, Sakmann and Neher, 1983, supra). Variations of patch clamp techniques can be utilized such as whole-cell recording, inside-out recording, outside-out recording, and perforated patch recording as are known in the art.
In whole-cell recording, the cell membrane covering the electrode tip is believed to be ruptured by suction in order to establish an electrical connection (and a chemical pathway) between the cell interior and the electrode solution. Because electrode solution is in great excess compared to the amount of cytosol in the cell (about 10 μl vs. about 1 pl), changing ionic species in the electrode solution will create concentration gradients across the cell membrane, providing a means to control the direction and magnitude of the transmembrane ionic flow for a given receptor/ion-channel complex.
In inside-out and outside-out patch clamp configurations, the cytosolic environment is lost by excision of a membrane patch from the entire cell (see, e.g., Neher and Sakmann, 1976, supra; Sakmann and Neher, 1983, supra). To obtain an excision of a patch in both the inside-out and the outside-out configurations, the cells are preferably attached to the bottom of the cell dish or recording chamber. In the case of acutely isolated cells, for example, poly-L-lysine can be used to fix the cells to the bottom of the chamber.
The inside-out configuration allows exposure of the cytosolic side of the membrane to solution in the recording chamber. It is therefore a method of choice for studying gating properties of second-messenger activated ion-channels at the single-channel level. Thus, the effects of cytosolic signaling molecules or enzymatic activity on ion-channel function can be studied by means of this configuration. The outside out configuration, on the other hand, allows exposure of the extracellular side of the patch. It can therefore be used to monitor the activity of ligand-gated or receptor-operated ion-channels.
One frequently used modification of the whole-cell configuration, the perforated patch mode also can be used (see, e.g., as described in Pusch and Neher, 1988, supra). In this technique, holes are selectively made in the cell membrane using a pore-building protein, such as amphotericin or nystatin (see, e.g., Akaike et al., 1994, Jpn. J. Physiol. 44: 433-473; Falke, et al., 1989, FEBS Lett. 251: 167; Bolard, et al., 1991, Biochemistry 30: 5707-5715) to create increased conductivity across the patched cell membrane without the loss of intracellular signaling molecules. In addition to measuring ion currents across ion channels at constant membrane potential, the patch clamp technique can be used to measure membrane voltage at a known constant or time-varying current. And in another aspect, the patch clamp technique can be used to monitor capacitance changes in cell membranes by providing a cell-based biosensor in the open volume reservoir and measuring impedance of the membrane across the membrane of the biosensor in an AC mode.
Patch clamp is traditionally performed using tapered glass micropipettes. However, recently there has been considerable effort in developing patch clamp devices on solid substrates such as silicon chips. Typically, these substrates have been equipped with one or several openings for placement and sealing of cells equivalent to the opening of a traditional patch clamp electrode. For example, Klemic, et al., in WO 01/59447, describe a planar patch clamp electrode array comprising a plurality of electrodes for performing patch clamp recordings on a plurality of patch-clamped cells.
Low noise levels provide better signal-to-noise ratios in patch clamp recordings. The low noise property of patch clamp is achieved by tightly sealing a glass microelectrode or patch clamp pipette onto the plasma membrane of an intact cell thereby producing an isolated patch. The electrical resistance between the pipette and the plasma membrane is critical to minimize background noise and should be in excess of 109 ohm to form a “giga seal”. The exact mechanism behind the formation of the “giga seal” is debated, but it has been suggested that various interactions such as salt-bridges, electrostatic interactions, and van der Waal forces mediate the interaction between the glass surface of the pipette and the hydrophilic heads in the lipid layer of the cell membrane (see, e.g., Corey and Stevens, 1983, In Single-Channel Recording, pp. 53-68, Eds. B. Sakmann and E. Neher. New York and London, Plenum Press). Under optimal conditions, single-channel currents in the higher femto-ampere (10−15 A) range can be resolved. Strategies to decrease noise (e.g., such as caused by a bad seal between the electrode and the cell) to facilitate formation of GΩ-seals include, but are not limited to, fire polishing of the glass electrode or treating the surface of the glass electrode using agents such as sigmacote. Dielectric noise and capacitive-resistive charging noise also can be decreased by selecting an expedient electrode/pipette geometry, using quartz glass, and by coating of the glass surface of the pipette with Sylgard® (silicone, PDMS) in order to decrease the capacitance of the pipette as much as possible.
However, it has proven difficult to obtain and maintain cells attached to both solid substrate chips and traditional path clamp micropipettes with good electrical sealing properties. Typical success rates for obtaining a whole cell-recording configuration with both techniques is about 50%. Further, the time periods during which cells can be held in a satisfactory position relative to a patch clamp micropipette to obtain a recording rarely exceeds 20 minutes.